Purification of blood coagulation factors
First Claim
1. A method for purifying a sample of human Factor IX, comprising:
- (a) loading a sample comprising different species of human Factor IX onto an immunoaffinity chromatography material comprising a gamma-carboxyglutamic-directed antibody (Gla-directed antibody);
wherein the amino acid sequence of the Gla-directed antibody comprises SEQ ID NO;
1 and SEQ ID NO;
2;
wherein the different species of human Factor IX comprise different numbers of Gla residues;
(b) eluting the sample of human Factor IX; and
(c) selecting a fraction obtained from the elution comprising an increase in the proportion of one or both of #1-11-Gla and #1-12-Gla species of human Factor IX compared with the proportion of one or both of #1-11-Gla and #1-12-Gla species of human Factor IX in the sample being purified;
wherein the total concentration of human Factor IX within the sample exceeds the binding ability of the immunoaffinity chromatography material.
3 Assignments
0 Petitions
Accused Products
Abstract
The present invention relates to the purification of vitamin K-dependent blood coagulation factors, such as Factor IX (FIX). In particular, the invention provides a method for purifying Factor IX having a desired content of gamma-carboxyglutamic acid from a sample comprising a mixture of species of said Factor IX having different contents of gamma-carboxyglutamic acid, said method comprising the steps of: (a) loading said Factor IX sample onto an immunoaffinity chromatography material coupled to a binding moiety for gamma-carboxyglutamic acid; (b) eluting said Factor IX; and (c) selecting a fraction obtained from said elution wherein the polypeptides in the fraction have the desired content of gamma-carboxyglutamic acids; characterized in that the total concentration of Factor IX within said sample exceeds the binding ability of the immunoaffinity chromatography material.
-
Citations
19 Claims
-
1. A method for purifying a sample of human Factor IX, comprising:
-
(a) loading a sample comprising different species of human Factor IX onto an immunoaffinity chromatography material comprising a gamma-carboxyglutamic-directed antibody (Gla-directed antibody); wherein the amino acid sequence of the Gla-directed antibody comprises SEQ ID NO;
1 and SEQ ID NO;
2;wherein the different species of human Factor IX comprise different numbers of Gla residues; (b) eluting the sample of human Factor IX; and (c) selecting a fraction obtained from the elution comprising an increase in the proportion of one or both of #1-11-Gla and #1-12-Gla species of human Factor IX compared with the proportion of one or both of #1-11-Gla and #1-12-Gla species of human Factor IX in the sample being purified; wherein the total concentration of human Factor IX within the sample exceeds the binding ability of the immunoaffinity chromatography material. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, 17, 18, 19)
-
-
14. A method for purifying a sample of human Factor IX, comprising:
-
loading a sample comprising different species of human Factor IX onto an immunoaffinity chromatography material comprising a gamma-carboxyglutamic-directed antibody (Gla-directed antibody); wherein the amino acid sequence of the Gla-directed antibody comprises SEQ ID NO;
1 and SEQ ID NO;
2;wherein the total concentration of human Factor IX within the sample exceeds the binding ability of the immunoaffinity chromatography material; b) eluting the sample of human Factor IX; c) selecting a fraction obtained from the elution as a second sample of human Factor IX; d) loading the second sample of human Factor IX onto an anion exchange chromatography material; e) eluting the second sample of human Factor IX; and f) selecting a fraction obtained from the elution of the second sample comprising an increase in the proportion of one or both high Gla species #1-11-Gla and #1-12-Gla of human Factor IX compared with the proportion of one or both of #1-11-Gla and #1-12-Gla species of human Factor IX in the sample being purified.
-
Specification